Ptprk immunogenic peptide

ABSTRACT

An immunogenic peptide isolated from PTPRK protein represents a novel HLA II restricted epitope recognized by CD4+T cells and is used in cancer immunotherapy or diagnosis.

The present invention relates to immunogenic peptides isolated from theprotein PTPRK (Receptor-Like Protein Tyrosine Phosphatase Kappa) and theuse thereof in the diagnosis and preventive or therapeutic treatment oftumors. More specifically, the invention provides a novel HLA-Class IIrestricted epitope recognized by CD4+ T cells and its use in thediagnosis, prevention or immune therapy of patients with melanomaexpressing PTPRK.

BACKGROUND OF THE INVENTION

The identification of tumor associated antigens able to induce aspecific anti-tumor T cell response has provided a new immunologicalapproach for the treatment of tumors. The large majority oftumor-associated antigens that have been up to now defined arerecognized by HLA-Class I restricted cytotoxic T lymphocytes (CTL),despite the crucial role played by CD4+ T cells which recognizeHLA-Class II antigens for the generation and maintenance of an effectiveimmune response in viral infection as well as in cancer. Lack oftumor-specific epitopes able to evoke a T cell-mediated helper responseand the self-nature of the majority of HLA-class I restrictedtumor-associated antigens constitute the major factors limiting thetherapeutic efficacy of vaccination trials in cancer patients.Therefore, the characterization of novel MHC class II-restrictedtumor-specific antigens appears of primary importance in order toprovide new and more efficient peptide-based vaccines. The mechanisms ofclass II MHC antigen processing/presentation pathway appear rathercomplex and not completely understood. Technical difficulties havehampered the discovery of class II HLA-restricted tumor antigens, thuslimiting the availability of known tumor-specific helper epitopes formelanoma, the most immunogenic among the human tumors.

PTPRK belongs to the family of the receptor-like plasmamembrane-spanning PTP molecules, and it is characterized by the presencein its extracellular portion of fibronectin type III repeats,immunoglobulin and meprin/A5/μ (MAM) domains (30, 31). PTPRK isexpressed in normal tissues such as spleen, prostate, ovary, andkeratinocyte epidermal cell lines but not in PBL and hematological celllines (24). Though the precise physiological role of PTPRK is stillunclear, its structural features and the ability to mediate homophilicinteraction among cells together with the observation that theexpression of PTPRK is induced by cell density strongly suggest acrucial role of this protein in the regulation of cell-cell contactformation (30). Moreover, it has been recently demonstrated that inhumans PTPRK co-localizes and is associated with β- and γ-catenin at thecell-cell contact area of adjacent cells (25). These findings stronglysupport the involvement of this protein in negatively regulating theaction of tyrosine kinase-induced events at cell junctions, possiblythrough the dephosphorilation of β- and γ-catenin. These functionalroles, and the finding that human PTPRK gene has been mapped to theputative tumor suppressor gene region 6q22.2-q22.3 (31, 32), which isfrequently deleted in melanomas (33, 34), overall support the hypothesisthat loss of expression as well as mutations occurring in functionaldomains of the PTPRK protein, could directly affect the phenotype ofnormal growing cells and play a role in tumor transformation andprogression.

DISCLOSURE OF THE INVENTION

With the aim of identifying HLA-class II melanoma antigens, we studiedthe CD4+ anti-tumor response in a melanoma patient who experienced afavorable prognosis being disease free 9 years after surgical resectionof a lymph node metastasis. This positive clinical evolution wasassociated with a strong and persistent CD8 mediated anti-tumor responsedirected against the differentiation antigen gp10 and TRP-2. Since CD4 Tcells are known to be essential for the generation and maintenance ofCD8 T cells, we investigated the possibility that the patient haddeveloped also a tumor-specific CD4-mediated response.

T lymphocytes obtained from tumor infiltrated lymph nodes wererepeatedly stimulated in vitro with the autologous tumor and T cellclones were generated by limiting dilution. CD4+ T cells clonesrecognizing the autologous tumor in an HLA-DR restricted fashion wereobtained, characterized in vitro for their fine specificity and used ascellular probe in a genetic approach aimed at defining the molecularnature of the recognized antigen. The screening of a cDNA expressionlibrary constructed using as template the RNA of the autologous melanomaled to the identification of the tyrosine phosphates receptor K gene(R-PTP-K) as encoding the antigen recognized by the CD4+ melanomaspecific clones. The R-PTP-K mRNA cloned by melanoma cells contains anon-conservative Gly→Arg mutation in the fourth fibronectin III-likedomain of the protein. This amino acid change generates a T cellsepitope presented by the HLA-DRβ1*1001 that is recognized by the CD4 Tcell clone used to screen the tumor cDNA library and by all the 5different clones isolated from the tumor infiltrated lymph nodes of thesame patient. The antigenic epitope was identified in the region 667-682of PTPRK_(Gly677→Arg682) and it has sequence PYYFAAELPPRNLPEP (SEQ ID N.1).

A first aspect of the invention is directed to the immungenic peptide ofSEQ ID N. 1 and the use thereof in the generation of antibodies and/or Thelper or cytotoxic cells, more generally in the induction of atumor-specific immune response, for diagnostic or therapeuticalapplications, in particular for the diagnosis, prevention or immunetherapy of tumors expressing PTPRK_(Gly677→Arg682).

The peptide of the invention may be prepared following differentprocedures. For example, it can be synthesized in solution or solidphase according to conventional techniques, or using an automaticsynthesizer. The theory and practice of peptide synthesis are known toany skilled person, see for example Stewart and Young (1984) Solid PhasePeptide Synthesis, 2^(nd) ed., Pierce Chemical Co.; Tam et al., J. Am.Chem. Soc., (1983) 105: 6442; Merrifield, The Peptides, Gross andMeinenhofer eds. Academic Press (1979) New York, pp. 1-284, hereinincorporated by reference.

For use in therapy, the peptide will be suitably formulated togetherwith pharmaceutically acceptable excipients. Suitable formulations forthe preventive or therapeutical treatment can be administered orally orparenterally, preferably subcutaneously or intramuscularly, and theywill contain an effective amount of the peptide. Said amount should beable to elicit a humoral or cell-mediated immune response directedagainst the tumor and will vary depending on the general conditions ofthe patient, the progression of the disease and other factors.

Preferably, the peptide of the invention is administered in the form ofa vaccine either for preventive treatment of melanoma-susceptibleindividuals or for the therapeutic treatment of melanoma patients. Thevaccine can be administered according to a single- or multiple- dosagescheme, at suitable doses and at different time intervals so as tomaintain or enhance the immune response. The peptide immunogenicity canbe increased by cross-linking or coupling with immunogenic carriers, orby use of suitable adjuvants.

Furthermore, the peptide may be conjugated with lipids, glucosideresidues or other peptides in order to increase bioavailability or theaffinity to HLA molecules.

In a further embodiment, the invention provides polyclonal or monoclonalantibodies, fragments or derivatives thereof such as Fab, Fv or scfv,able to recognize and bind the peptide SEQ ID N. 1. The isolatedantibodies can be used in tumor immune therapy or in immune diagnostictechniques for the definition of tumors expressingPTPRK_(Gly677→Arg682). In a yet further embodiment, the inventionprovides isolated CD4+ T cells specifically recognizing a tumorexpressing PTPRK_(Gly677→Arg682) and the use thereof for inducing acell-mediate immune response against such tumor. These cells can beisolated from PBMC obtained from the patient to be subjected to thetreatment, and they can be activated in vitro with the peptide SEQ ID N.1, optionally in the presence of cytokines, or using cells carrying thepeptide in association with HLA-Class II molecules, such as APC (antigenpresenting cells) expressing the allele HLA-DR β1*1001 loaded with thepeptide. APCs can be genetically modified, e.g. by transfection with aviral or retroviral vector, so as to express the specific allele. HLA orthe peptide or a precursor thereof. Modified HLA cells can be used toactivate T cells either in vitro or in vivo. In vitro activated T cellscan be subsequently reintroduced in the patient to prevent-the onset, toarrest the growth or to reduce the amount of tumor cells. Before beingreintroduced into the patient, lymphocytes may be purified, for exampleby means of an affinity column using an antibody directed against CD4 orother markers.

In a further embodiment the invention provides an isolated nucleic acidmolecule encoding the epitope of PTPRK_(Gly677→Arg682) herein described,preferably the sequence (SEQ ID N. 2)CCGTATTACTTTGCTGCAGAACTCCCCCCGAGAAACCTACCTGAGCCTas well as a vector and a host cell including said sequence. DNAmolecules containing the peptide-encoding sequence, or a part thereof,and the gene constructs thereof can be used in the vaccination ofsubjects at risk of developing tumors, particularly melanoma, or cancerpatients. DNA immunization can be carried out according to knowntechniques (Donnelly J. J. et al., 1994, The Immunologist 2: 1). Theintramuscular administration route is referred, but also the parenteraland mucosal routes can be used (pnas 1986, 83, 9551; wo90/11092).Moreover, DNA can be adsorbed onto gold particles for the subcutaneousadministration with a biolistic apparatus (Johnston, 1992 Nature, 356,152).

In addition to the above described therapeutic uses, nucleic acidmolecules containing the peptide-encoding sequence, or a part thereof,as well as the peptide itself, can be used in the diagnosis of melanomaexpressing PTPRK_(Gly677→Arg)682, for instance by PCR analysis orimmunoassays using epitope-specific antibodies. Furthermore, complexesbetween the peptide SEQ ID N. 1 and HLA-DR β1*1001 cells can be used formonitoring in vitro or ex vivo the immune response of subjectsvaccinated with the peptide.

DESCRIPTION OF THE FIGURES

FIG. 1

Functional characterization of anti-melanoma T lymphocyte clone TB515.(A) Sensitivity of ⁵¹Cr-labeled Me15392 and LCL15392 cells to lysis byTB515 clone: ⁵¹Cr-release was measured after 5 h. The assay wasperformed in the absence (▪) or in the presence (▾) of the anti-HLA-DRAb, L243. (B) Cytokines release assay: TB515 clone was incubatedovernight with autologous tumor or 15392LCL at 1:1 cell ratio in thepresence or absence of the L243. Supernatant was collected, and thecontent of INF-γ, GM-CSF, TNF-α and IL-2 was evaluated by commerciallyavailable ELISA assay. S.D.≦5%.

FIG. 2

Complementary DNA of clone #11 encodes for the TBS15 recognized antigen.(A) CIITA ⁺293 were transfected with cDNA#11 in pEAK8.5-Ii alone ortogether with pcDNA3.1 -DRB1*0102 or pcDNA3.1-DRB1*1001, respectively.CIITA ⁺293 singly transfected with pcDNA3.1-DRB1*0102 orpcDNA3-DRB1*1001, and CIITA ⁺293 co-transfected with recombinant pcDNA3encoding green fluorescent protein (GFP) and pcDNA3.1-DRB1*1001 wereused as negative control. (B) cDNA#1 was subcloned in pcDNA3.1 andcotransfected with pcDNA3.1-DRB1*1001 in CIITA ⁺293. Clone TB515(1×10⁵lymphocytes/well) was added to each transfectant and after 24 h,supernatants were collected and the content of IFN-γ evaluated by ELISA.In both panels, Mel5392 was used as positive control. Transfectats wereall evaluated for the ability to induce IFN-γ release by TB515.

FIG. 3

(A) PTPRK mRNA expression in Me15392 cells. 10 μg of poly(A)⁺mRNAobtained from Me15392 were analyzed by Northern blot. Hybridization wasperformed using an equimolar mixture of three ³²P labeled probesspanning the extracellular, the transmembrane and the intracellularregions of the PTPRK cDNA (See Materials and Methods for details). Laneswere loaded with a comparable amount of mRNA as checked by β-actinhousekeeping gene hybridization (data not shown). Samples: Me15392, mRNAobtained from cultured 15392 melanoma cells; PBL, pool of mRNA from 4healthy donors; A431, mRNA from epidermoid tumor cell line.

(B) RT-PCR analysis of PTPRK mRNA expression profile. The regionspanning the intracellular phosphatase domain was amplified by theprimers F3/Rδ that generate a specific amplification band of 650 bp. Thereaction conditions were set within the linear range of DNAamplification. The amount of template was adjusted in order to obtaincomparable levels of β-actin, thus allowing direct comparison ofamplified PTPRK among the examined samples. As a representative example,evaluation of PTPRK expression by RT-PCR analysis is reported for 5 outof 10 melanoma cell lines examined: 4 metastatic (Me15392, Me335, Me337and Me349) and one primary (Me366) melanoma cell lines. A PBL pool of 4healthy donors (PBL), and normal melanocytes (FM2093) are also included.

Picture is representative of 3 independent experiments.

FIG. 4

Characterization of cDNA #11. cDNA #11 and related minigenes arerepresented as boxes aligned to a schematic structure of PTPRK protein.Black square in each minigene indicates the position of an ATG codon inframe with the starting ATG of the full-length gene (GenBankNM_(—)002844). The mutated nucleotide (g→a) occurring at position 2249is indicated. Minigenes were synthesized by PCR amplification of cDNA#11 using an identical forward primer (F2) coupled with different,nested reverse primers mapping downstream the mutation (EPR1, EPR2,EPR2WT, and EPR3 reverse primers, indicated by the arrows). Minigeneswere cloned into expression vector pcDNA3/TOPO and then co-transfectedwith pcDNA3-DRB1*1001 or pcDNA3-DRB1*0102 into CIITA⁺-293 cells. CloneTB515 (1×10⁵ cells/well) was added to each transfectant, and after 24 hsupernatants were evaluated for the content of IFN-γ by ELISA. In thetable: +, positive recognition by TB515; −, no recognition by TB515.EP2wt minigene contained the non-mutated (g) nucleotide. Amino acidsequence in the bottom of the figure was deduced from the sequencing ofcDNA #11. Abbreviations in the figure: LS, leader sequence; MAM,meprin/A5/R-PTPμ motif; Ig, immunoglobulin-like domain; FNIII,fibronectin type III-like domain; TM, transmembrane; PTP,protein-tyrosine phosphatase domain; R, arginine deriving from thenucleotide g→a mutation.

FIG. 5

Identification of the TB515 epitope. (A, B, C) LCL15392 cells (5.0×10³cells/well) pulsed for 2 h at 37° C. with the synthetic peptides indifferent concentrations were used to stimulate TB515 T lymphocytes. TheTB515 (5×10³ cells/well) was added, and after 18 h medium was collectedand IFN-γ measured by ELISA. Identical results were obtained usingLCL3700, sharing only the DRB*1001 with pt15392 (not shown). (D) 5×10³LCL15392 cells were incubated with various concentrations of thecompetitor peptides for 15 min. Competitor peptides included:PYGFAAELPPRNLPEP, modified in position 3, the wild typePYYFAAELPPGNLPEP, and the HLA-A3 binding peptide ILRGSVAHK which wasused as negative control. PYYFAAELPPRNLPEP peptide was then added at 100nM. After 1 hr of additional incubation at 37° C., TB515 (5×10³cells/well) was added, and after 18 h medium was collected and IFN-Ymeasured by ELISA. Identical results were obtained using LCL3700 sharingonly the DRB*1001 with pt15392. Mutated amino acid is written in bold;substituted amino acids are underlined.

FIG. 6

Peptide specificity of T lymphocyte clone TB48. LCL15392 cells (5.0×10³cells/well) pulsed for 2 h at 37° C. with the synthetic peptides indifferent concentrations were used to stimulate TB48. The TB48 clone(5×10³ cells/well) was added, and after 18 h medium was collected andIFN-γ measured by ELISA. Identical results were obtained using LCL3700,sharing only the DRB 1001 with pt15392.

FIG. 7

PTPRK epitope specific immunity in PBMCs of pt 15392. PBMCs of pt15392,obtained during the disease-free period 12 months after surgery, werestimulated in vitro with the mutated PYYFAAELPPRNLPEP peptide. At theend of the third week of culture, the generated T cell lines wereevaluated by the ELISPOT assay for the capacity to release IFN-γ inresponse to peptide or autologous tumor stimulation, in the presence orin the absence of the anti-HLA-DR Ab L243. T cells were incubated with(1) medium, (2) LCL15392, (3) LCL15392 pulsed with 4 μg ofPYYFAAELPPRNLPEP, (4) LCL15392 pulsed with 4 μg of PYYFAAELPPRNLPEP andincubated with 10μg/ml of anti-HLA-DR L243 Ab, (5) LCL15392 pulsed with2 μg of PYYFAAELPPRNLPEP, (6) LCL 15392 pulsed with 2 μg ofPYYFAAELPPRNLPEP and incubated with 10 μg/ml of anti-HLA DR Ab L243, (7)LCL15392 pulsed with 4 μg of the wild type PYYFAAELPPGNLPEP peptide, (8)autologous Me15392, (9) autologous Me15392 incubated with anti-HLA DR AbL243.

Abbreviations: pt15392, patient 15392; LN, lymph node, gp100,glyco-protein 100; TRP-2, tyrosinase-related protein 2; PTPRK,receptor-like protein tyrosine phosphatase kappa; RT-PCR, reversetranscribed polymerase chain reaction; CIITA, Class II Transactivator;Ii, invariant chain; GFP, green fluorescent protein; LCL, lymphoblastoidEBV-trasformed B cells line, MTS, melanosomal transport signal; MAM,meprin/A5/PTPRμ motif; E, effector cell; T, target cell; TCR, T cellreceptor.

Materials and Methods

Cell lines. The clinical course of patient (pt) 15392 (HLA-A*0301,B*40012, B*1402, C*0602, C*8002, DRB1*0102, DRB1*1001), and the in vitrostabilization of the melanoma cell line Me15392 have been alreadydescribed (16). By cell surface analysis Me15392 cells were shown to bepositive for class I HLA and to constitutively express DR, DP, but notDQ class II HLA. LCL15392 and LCL3700 are EBV-trasformed B cell linesobtained from peripheral blood mononuclear cells (PBMCs) of pt15392 andof an healthy donor, respectively. LCL3700 shares with pt15392 theDRB1*1001 only. 293-EBNA cells (wt293) (Invitrogen, CA 9200, USA) andClass II Transactivator⁺ 293-EBNA cells (CIITA ⁺293) were maintained inDMEM medium (Euroclone, Europe, TQ4 5ND Devon, UK) with 10% FCS. CIITA⁺293 cells were obtained by trasducing wt293 cells with CIITA-encodingretroviral vector. CIITA ⁺293 cells were immunoselected using L243, amAb specific for HLA-DR alleles.

Generation of tumor-specific T cell clones. Tumor infiltrating T reverse5′-ATTAGGACAAGGCTGGTGGGCACT-3′). The non-conservative point mutation(g→a) was confirmed either by sequencing both DNA strands of amplifiedproducts from reverse-transcribed Me15392 poly(A)⁺RNA (forward primer F25′-GTGCTCCTATCAGTGCTTAT-3′, reverse primer R2 5′-GCGTACGCACTGGGTTTT-3′)or from Me15392 genomic DNA (forward primer5′-CTGCACCCACACCGAACCAAGAGAGAA-3′, reverse primer5′-CGCCTGGAAATAGATGTTGTATCCTTT-3′).

Mini-genes were prepared from cDNA #11 as PCR amplification products ofdifferent lengths. All the amplicons were obtained using the same senseprimer F2 coupled with four different antisense primers: EPR15′-CCGATTGTCACCCACAGTGAA-3′, EPR2 5′-GGGCAGGCTCAGGTA-3′, EPR35′-CTCGGGGGGAGTTCT-3′, and EPR2WT 5′-GGGCAGGCTCAGGTAGGTTTCCCG-3′. Thelatter was used for the preparation of the EP2wt minigene containing thewild type nucleotide (underlined in the EPR2WT primer). PCR productswere cloned into pcDNA3.1/V5-His TOPO vector.

The Ii-EP2wt fusion minigene was obtained by excision of EP2wt minigenefrom TOPO vector with AscI and XbaI enzymes, followed by cloning theinsert into the AscI and XbaI sites of pEAK8.5/Ii vector. This reactiongave rise to an in frame fusion construct.

Northern Blot and RT-PCR analysis of R-PTPK expression. Poly(A)+ RNAsfrom Me15392, allogenic melanomas and PBL lines were isolated asdescribed above. Total RNA was isolated by using RNAqueous™-4PCR kit(Ambion, Austin, Tex., 78744, USA). For Northern blot experiments, 10 μgof each RNA sample was subjected to electrophoresis in a 1% formaldehydeagarose gel and transferred to a nylon membrane (Hybond-N+, AmershamBiosciences, Inc. Piscataway, N.J. 08855-1327, USA). The probes werelabeled with [alpha-³²P]CTP by the random priming method (AmershamBiosciences), and pre-hybridization and hybridization were performedaccording to the Hybond-N+ paper guidelines. Membranes were washed fourtimes with serially diluted solutions of SSC (from 0.03M to 0.0015M).Probes A and C were obtained by PCR amplification of Me15392 poly(A)⁺RNA with specific primers. Probe A, specific for the 5′ region of thegene (bases 241-1110 of the gene), was synthesized with primers forwardF1 (5′-GGCGCTGCCTGCTTTTGT-3′) and reverse R1 (5′-GGAGGAGCAATGGGTCTT-3′).Probe C, specific for the region encoding the two intracellularphosphatase domains (bases 2925-4547 of the gene), was derived withprimers forward F3 (5′-CTTGGGATGTAGCTAAAAAAGATCAAAATA-3′) and reverseSTOP (5′-CCAACTAAGATGATTCCAGGTACTCCAA-3′). All the amplificationproducts were sequenced before being used as probes. DNA clone #11(bases 2084-2751 of the gene) was used as probe B.

The RT-PCR analyses of PTPRK expression-profile in normal and tumorscell lines were performed with the forward primer F3 and with thereverse primer Rδ (5′-CACCCTCTCTTTCAGCCAT-3′) under the followingconditions: 2′ 94° C., 34 cycles consisting of 1′ 94° C., 2′ 54° C., 3′72° C., and finally 10′ 72° C. Conditions were set in order to obtainlinear DNA amplification. The amplified DNAs were loaded on agarosegels, stained with ethidium bromide, and analyzed with a dedicatedsoftware (Image Master VDL-CS, Amersham Pharmacia Biosciences). Standarddeviations were ≦5% on triplicate experiments. The level of expressionof each sample was normalized for RNA integrity by taking into accountthe level of expression of the β-actine gene (reaction conditions: 4′94° C., 21 cycles consisting of 1′ 94° C., 2′ 68° C., 2′ 72° C., andfinally 10′ 72° C., corresponding to linear DNA amplification).

Peptides synthesis. Peptides were synthesized by conventional solidphase peptide synthesis, using Fmoc for transient NH₂-terminalprotection, and characterized by mass spectrometry. All the peptidesused were >⁹⁵% pure (Neosystem, Strasbourg, France). Peptides weredissolved at 5 mg/mL in DMSO, stored at −20° C., and diluted in RPMImedium supplemented with 10% human serum immediately before use.

Epitope reconstitution assay. To analyze peptide recognition, 5×10³LCL15392 or LCL3700 cells were seeded in 96 microwells in 100 μl of RPMI1640-10% PHS and then pulsed with different concentrations of therelevant peptide. Peptide loading was allowed to proceed for 2 h at 37°C. before effector cells were added to give a final E:T ratio of 1:1.Supernatants were collected after 18 h and IFN-γ content was determinedby ELISA (Mabtech AB, Stockholm, Sweden). Competition experiments wereperformed incubating 5×10 LCL15392 or 5×10 LCL3700 with variousconcentrations of the competitor peptides for 15 min before the additionof the PYYFAAELPPRNLPEP peptide at 100 nM. After 1 hr of additionalincubation at 37° C., T cell clone was added at the final ratio of 1:1.

Generation of PTPRK specific T cells from pt15392 PBMCs. PBMCs ofpt15392 obtained at 12 months after surgery, during the disease-freeperiod of follow up, were stimulated in vitro with PYYFAAELPPRNLPEPpeptide as previously described (15). Briefly, PBMCs (2×10⁶/well) wereweekly stimulated with autologous peptide-pulsed monocytes. At the endof each stimulation, peptide-specific reactivity was monitored byElispot assay.

IFNγ-Elispot assay. 96-well nitrocellulose plates (Millititer,Millipore, Bredford, Mass.) were coated overnight with 50 μl/well of 8μg/ml anti-human IFN-γ mAb (Mabtech). Wells were then washed and blockedwith Iscove's modified DMEM (BioWhittaker) and 10% human AB-serum for 2h at 37° C. T cells (2×10³ or 2×10⁴) were mixed with 1.5×10⁴peptide-pulsed autologous LCL cells and then seeded in the 96 pre-coatedwells. T cells incubated with medium alone or with pokeweed mitogenserved as negative and positive controls, respectively. After 24 h ofincubation at 37° C. and 5% CO₂, Elispot was then performed according tomanufactory instructions. Briefly, plates were washed six times withPBS+0.05% Tween-20. Wells were incubated for 2 h at 37° C. with 50μl/well of biotinylated mouse anti-human IFN-γ mAb (Mabtech,) at aconcentration of 2.5 μg/ml. After washing four times with PBS, 100 μlstreptavidine-alkaline phosphatase (150 μg/ml) diluted 1/1000 was addedfor 2 h at room temperature. After another washing step with PBS, 100μl/well of BCIP/NBT substrate (BioRad, CA, 94547,USA) was added to eachwell for 10-20 min. Color development was stopped by washing underrunning tap water. After drying at room temperature, IFNγ secreting Tcells were counted using the automated image analysis system ElispotReader (AID, Strassberg, Germany). Each experiment was performed intriplicate.

Results

Tumor-specific CD4⁺ T cell clones. 40 tumor-specific CD4+T cell clones(15392CD4⁺T) were obtained by limiting dilution cloning of TIL isolatedfrom a LN metastasis of pt15392, and stimulated in vitro with theautologous tumor for two weeks. TCR analysis by RT-PCR revealed that allclones could be grouped in 5 different subsets according to the TCRAVand TCRBV combination (Table). Functional studies were performed on asingle T cell clone representative of each subset. All the clones werefound to display identical functional activity in response to tumorstimulation. Data reported in FIG. 1 were obtained with clone TB515 andare representative for all the T cell clones tested. Upon stimulationwith the autologous melanoma, all the clones exerted an HLA-DRrestricted lytic activity against Me15392 cells, since the lysisdetected by a ⁵¹Cr release assay, was strongly inhibited in the presenceof L243, a mAb directed to a monomorphic determinant of HLA-DR (FIG.1A). Moreover, upon tumor stimulation the tumor-specific CD4⁺ T cellclones also released a cytokine cocktail compatible with Th1 profile,including INF-γ, GM-CSF, TNF-α and IL-2, but not IL-4 and TGFβ (FIG.1B).

Mutated PTPRK gene encodes the TB515 epitope. Due to its in vitro growthcapacity, TB515 was further selected and used to molecularlycharacterize the melanoma specific antigen. The genetic approachemployed for this purpose was developed as an optimization of recentreported methodologies used for the identification of tumor antigens(18-20). The 293-EBNA1 cells stably transfected with CIITA cDNA (CIITA⁺293) (21) were used as recipients for the cDNA tumor library. FACSanalysis with anti-HLA-DR Ab revealed a significant increase in theexpression of class II HLA on CIITA ⁺293 cells (not shown). To force theentrance of the tumor-derived proteins into the class II HLA processingpathway, and thus potentially improving the sensitivity of our screeningmethod, we constructed a chimeric invariant chain (Ii)/tumor cDNAlibrary (22). The cDNA library divided into pools was then transfectedinto CIITA ⁺293 cells together with 100 ng of plasmid containing theDRB1*0102 or DRB1*1001 alleles cloned from the same patient.

The screening of transfectants led to the isolation of one positive CDNAclone (cDNA #11) that was recognized by TB515 T cells whenco-transfected into CIITA+293 with DRB1*1001 (CIITA⁺-DRB10⁺293) but notwhen co-transfected with the DRB1*0102 allele (CIITA⁺-DRB1⁺293) (FIG.2A). No recognition occurred when cDNA #11 and DRB1*1001 weretransfected into 293 not modified by CIITA (wt293), thus furtherconfirming the requirement of an active Class II-HLA processingmachinery for TB515 activation.

DNA sequencing analysis indicated that cDNA #11 was homologous to apartial region of the Receptor-Like Protein-Tyrosine Phosphatase Kappa(PTPRK, GenBank NM_(—)002844), a type II tyrosine phosphatase (PTPs),which is one of the five subfamilies of transmembrane receptor-like PTPs(23). Tumor derived cDNA #11 extended from 2084bp to 2751 bp of thepublished NM_(—)002844 PTPRK cDNA sequence, encompassing the regioncoding for the fourth C-most extracellular fibronectin III-like domain,the whole transmembrane domain, and the very initial intracellularsequence. The clone presented a non-conservative g→a point mutation atnucleotide 2249 in the fourth fibronectin III-like domain of the gene(NM 002844), that led to a Glycine-to-Arginine substitution (G→R) in thecorresponding protein (the GenBank accession number for the sequence ofPTPRK cDNA derived from Me15392 is AF533875).

In order to confirm that this amino acid change did not account for asingle nucleotide polymorphism, but represented a somatic mutationoccurring in tumor tissue, the region encompassing the mutation wasamplified and sequenced starting either from genomic DNA or cDNAobtained from both PBL and LCL of pt15392. None of the analyzed samplesshowed the g→a transition and only the wild type sequence was found(data not shown). Moreover, DNA and cDNA derived from 10 additionalmelanoma cell lines were similarly analyzed but none of them displayedany mutation at nucleotide 2249.

Invariant chain sequence is not necessary for the HLA-class IIpresentation of R-PTPK derived epitope. Sequence analysis of therecombinant plasmid containing the cDNA clone # 11 revealed that the Iichain translation was shifted in respect to the frame corresponding tothe proper translation of PTPRK protein. Therefore, we postulated theexistence in the recombinant plasmid of an additional open reading framedirected by an additional internal ATG. By analyzing the sequence ofcDNA#11, we found a Kozak-like sequence ((g/a)nnatgg) whose ATG(position 2165 in the GenBank NM_(—)002844 sequence) was in frame withthe authentic first starting methionine of the PTPRK gene. To verify theself-sufficient translation capacity of this inner ATG codon, cDNA #11was excised from pEAK8.5/Ii vector and inserted into the Ii-lackingexpression vector pcDNA3.1. The new recombinant plasmid was thenevaluated for the capacity to trigger the TB515 activation whentransfected into CIITA ⁺293 cells together with the DRB1*1001 (FIG. 2B).As expected, the cDNA #11 cloned in the absence of the Ii chain inducedINF-γ release by TB515 clone in a specific DRB1*1001-restricted fashion.The recognition was as efficient as that of cDNA #11 in pEAK8.5/Iivector or Me15392 tumor (FIG. 2B) indicating that, in the absence of Iistart codon, cDNA #11 could be translated from its own internal ATG and,moreover, that cDNA #11 could be naturally processed and presented in aHLA-class II pathway per se with no need for an Ii chain mediatedintracellular redirection.

Expression patterns of R-PTPK mRNA. To obtain more information about thePTPRK specific transcripts in Me15392, Northern blot analysis wasperformed (FIG. 3). To encompass the whole PTPRK mRNA, hybridization wascarried out using an equimolar mixture of three specific probes whichmapped in different regions of PTPRK gene. The examined samples alsoincluded PBL poly(A)⁺RNA, as a negative control of PTPRK expression, andtransformed epidermal cell line A431, as a positive control of thefull-length transcript since this cell line is known to have a highlevel of PTPRK gene expression (24). Melanocytes could not be analyzed,since no sufficient mRNA was obtained form the in vitro growing cells.As expected, no significant hybridization band was observed in PBL, thusconfirming previous data (24), while both A431 and Me15392 poly(A)⁺ RNAgave rise to a rather complex pattern (FIG. 3A). Although additionalspecific bands around 1800 bp and 1000 bp were clearly detectable, bothsamples presented an intense signal at about 5600 bp corresponding tothe full-length transcript. All together, these results indicate thatMe15392 may express a complete PTPRK protein, while the biologicalsignificance of the partial mRNA transcripts remains to be addressed.

The expression of PTPRK mRNA in other tumor and non-tumor cells wasstudied by RT-PCR. RT-PCR amplifications were carried out from total RNAusing the primers pair F3/Rδ, mapping in the intracellular, N mostphosphatase domain (amplification product length 650 bp). Primers weredesigned to be highly specific for PTPRK gene. Their specificity wasexperimentally confirmed, and amplification products obtained fromdifferent cell lines were sequenced and showed to correspond only toPTPRK, while no sequences of other R-PTP or PTP genes, even those withhigh homology to PTPRK, were found. Reaction conditions were set to havelinear DNA amplifications, and comparable amount of mRNA was used fromeach cell lines as evaluated by β-actin amplification. While clearlydetectable in melanocytes (FM2093 cell line), PTPRK was differentlyexpressed in 10 melanoma cell lines tested, 5 of which showed only abarely detectable band comparable to that obtained from PBL, that wasnegative in Northern blot analysis (FIG. 3A). Some representativeexamples are given in FIG. 3B.

Identification of PTPRK derived peptide reconstituting the epitope forTB515. To assess the importance of the G→R mutation for T cellrecognition, a wild type (not mutated) version of cDNA #11 wassynthesized by PCR reaction and co-transfected with DRB1*1001 cDNA intoCIITA ⁺293 cells. No recognition of the wild type sequence by thetumor-specific 15392CD4+ T clones was observed (data not shown), thussuggesting a direct role of the mutated amino acid in the epitopeformation. To further sharpen the epitope-coding region of cDNA #11, aseries of mini-genes were synthesized by PCR reactions of cDNA #11 usingan identical forward primer (F2) coupled with different nested reverseprimers mapping downstream the mutation (FIG. 4). The minigenes, whichall contained the ATG starting codon of cDNA#11, were cloned into theexpression vector pcDNA3/TOPO (Invitrogen), then transfected in CIITA⁺293 expressing either DRB1*0102 or DRB1*1001, and finally evaluated forTB515 recognition. The EP2 minigene was the shortest construct beingrecognized. As expected, the wild type version of the EP2 minigene(EP2WT), obtained using reverse primer EPR2WT bearing the wild typenucleotide (a/g mismatch) was not recognized by TB515 lymphocytes whencloned into the expression vector with or without the invariant chain.

These transfection experiments sharpened the potentially immunogenicamino acid region to a 26 amino acids-long peptide, shown in FIG. 4,that contained the mutation. To identify the TB515 epitope, severalhexadecamer overlapping peptides were synthesized that spanned theidentified 26 amino acid region. The peptides were evaluated indifferent doses for their ability to sensitize in vitro autologous LCLcells to recognition by the anti-melanoma TB515 clone. Among thehexadecamer peptides tested, PYYFAAELPPRNLPEP (PTPRK₍₆₆₇₋₆₈₂₎), whichextends from amino acid 667 to 682 of the protein and contains themutation (FIG. 5, in bold), was the one giving rise to the strongestINF-γ release by TB515 clone. Indeed, titration experiments showed thatthis peptide could be detected by TB515 cells at a concentration as lowas 10 nM, reaching its half-maximum effect at 100 nM (FIG. 5A). Thesubstitution in the immunogenic peptide of the mutated arginine residuewith the wild type glycine (FIG. 5A) completely abrogated stimulatoryability, thus definitely showing the relevance of the mutation for Tcell mediated recognition. Further experiments with peptidesprogressively losing an amino acid residue at either the 5′ or the 3′end (FIG. 5B and 5C) were aimed at identifying both the minimal epitopecore and candidate anchor amino acids. In particular, the omission orthe substitution with non relevant amino acids of Y₆₆₉ (FIG. 5B) or L₆₇₉and N₆₇₈ (FIG. 5C) strongly affected the capacity of the PTPRK₍₆₆₇₋₆₈₂₎peptide to stimulate TBS515 clone, thus suggesting a role for theseamino acids in position 3, 11 and 12 in the formation of theHLA-DR10/peptide/TCR complex. The wild type peptide PYYFAAELPPGNLPEP andthe PTPRK₍₆₆₇₋₆₈₂₎ with the Y₆₆₉ substituted by a G were both able tobind the HLA-DR10, since in a competition assay they efficientlyinhibited the recognition of the PTPR₍₆₆₇₋₆₈₂₎ peptide by clone TB515(FIG. 5D).

The other 4 T lymphocyte clones described as having different TCR wereindeed directed against the same epitope, they did not recognized thewild type peptide and all of them displayed a similar affinity for themutated peptide as that observed for TB515 (data not shown). Moreover,the PTPRK₍₆₆₇₋₆₈₂₎ peptide bearing a substitution at residue Y₆₆₉ wasefficiently recognized by TB48 clone, confirming that Y₆₆₉, althoughessential for the TB515 mediated recognition, did not cruciallyinfluence the HLA-DR10 binding capacity of PTPRK₍₆₆₇₋₆₈₂₎ peptide (FIG.6).

Pt15392 developed systemic immunity against the PTPRK derived, HLA-DR10presented epitope. To establish whether pt15392 also developed asystemic, epitope-specific T cell immunity, PBMCs obtained 12 monthsafter the surgical resection of the lymph node metastasis were culturedin vitro with PYYFAAELPPRNLPEP peptide at 1 μM. After 3 weeks of invitro stimulation with peptide-pulsed autologous monocytes, the culturedT cells showed a peptide-specific reactivity as detected by Elispotassay, indicating the presence in the peripheral blood ofpeptide-specific T cells (FIG. 7). These cells were also able torecognize the autologous tumor in an HLA-DR restricted fashion.Conversely, no specific reactivity was detectable in PBMCs obtained fromDRB1*1001 positive healthy donors. TABLE Functional characterization andTCR expression of tumor-specific CD4⁺T clones. Recognition of theautologous tumor ^(e)IFN-γ release ^(a)Subset ^(b)Clone ^(c)TCRAV/TCRBV^(d)Lysis (%) (pg/ml) T1 TB 515 13/21 50 (10) 1000 (200) T2 TB 426  3/1374 (35) 1200 (300) T3 TB 48   3/21 35 (15) 1100 (200) T4 TB 674 13/19 12 (5) 800 (100) T5 TB 89  17/21 41 (10) 1060 (200)^(a)Forty anti-melanoma 15392CD4⁺T cell clones were generated bylimiting dilution of 15392CD4⁺TIL stimulated in vitro with theautologous melanoma for two weeks. The 15392CD4⁺T clones were grouped in5 distinct subsets (T1-T5) expressing different TCRAV and TCRBV.^(b)For each subset a tumor-specific clone, further cultured andexpanded in vitro, is indicated.^(c)Analysis of TCR was performed by RT-PCR using a panel of TCR Vα andVβ specific primers (see Materials and Methods).^(d)The ability of the indicated clones to lyse the autologous tumor wastested in a 5h ⁵¹Cr-release assay at E:T ratio 30:1. Data are reportedas % of lysis. Numbers in parenthesis refer to % of lysis achieved inthe presence of the monoclonal anti- HLA-DR L343 mAb used at 10 μg/mlfinal concentration.^(e)Cytokine release assay was performed to evaluate the ability of theindicated T cell clones to recognize the autologous tumor (see Materialand Methods for details). The amount of IFN-γ released after tumorstimulation in the absence or in the presence (values in parenthesis) ofL343 used at 10 μg/ml final concentration is expressed in pg/ml.

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1. The PTPRK_(Gly)677→Arg682 immunogenic peptide of SEQ ID NO:
 1. 2. Amonoclonal or polyclonal antibody, or an active fragment thereof, whichselectively binds the peptide of claim
 1. 3. An isolated nucleic acidmolecule encoding the peptide of claim
 1. 4. An expression vectorcarrying the nucleic acid molecule of claim
 3. 5. A host cell containingthe vector of claim
 4. 6. An isolated CD4+ T lymphocyte able toselectively recognize and bind the peptide SEQ ID NO: 1 associated to aHLA-Class II molecule.
 7. A T lymphocyte according to claim 6, whichselectively recognizes and binds a peptide/HLA-DR β1*1001 complex. 8.Antigen presenting cells carrying the peptide SEQ ID NO: N. 1 bound to aHLA-DR β1*1001 molecule.
 9. Pharmaceutical composition containing thepeptide SEQ ID NO: 1 or a nucleic acid molecule encoding it, inadmixture with pharmaceutically acceptable excipients.
 10. Thepharmaceutical composition of claim 9, in the form of a vaccine.
 11. Amedicament for the preventive or therapeutic treatment of cancercomprising one of peptide SEQ ID NO: 1 and nucleic acid moleculesencoding it.
 12. The medicament claimed in claim 11, for the preventiveor therapeutic treatment of melanoma expressing PTPRK_(Gly677→Arg682).13. A diagnostic composition comprising peptide SEQ ID NO: 1 or of anucleic acid molecule encoding it.
 14. The composition according toclaim 13, wherein said diagnostic composition is utilized in thecharacterization of melanoma expressing PTPRK_(Gly) 677→Arg682.